Abstract. Protein interactions mediate cellular communication, signaling, and numerous other cell functions. Recognition of these interactions has inspired much of the activity in the field of drug discovery in the context of designing inhibitors to selectively bind and disrupt the functions of protein targets. The drug discovery process faces new challenges with the growing threat of drug-resistant strains of pathogens and the search for more specific therapeutics to fight cancer. Breakthroughs have been propelled by progress in the understanding of cell biochemistry as well as technological advances in biophysical methods that facilitate the characterization of protein interactions and structures of complexes. This proposal focuses on the development of ultraviolet photodissociation (UVPD) in conjunction with native-spray mass spectrometry as an innovative strategy for analysis of protein complexes. The identification and characterization of protein-ligand and protein-protein complexes offers the opportunity to provide insight into protein function at a molecular level and accelerate drug discovery. The objectives of this proposal include: Aim 1: Development of UVPD for examining protein-ligand complexes. We will develop robust native-spray/UVPD-MS strategies to create conformational maps of protein-ligand complexes governed by different binding modes. The classes of ligands will include substrates, cofactors, and inhibitors. Aim 2: Integrating separation methods with UVPD-MS for higher throughput analysis of protein complexes. To expand the versatility of the UVPD-MS strategy, we will adapt it for a higher throughput workflow via integration with size exclusion chromatography (SEC). Front-end SEC will make sample introduction reproducible and automated, thus making it better suited for quantitative applications. Aim 3. Extending UVPD-MS for protein-protein complexes. Many proteins exist and function as part of multimeric complexes, thus motivating our interest in extending UVPD-MS methods for characterization of protein-protein and protein-protein-ligand complexes. Aim 4: Application of UVPD-MS methods for key biological problems. The methods developed in Aims 1 ? 3 will be utilized to attack a number of high impact biological problems via collaborations with research groups in molecular biology, biochemistry, and medicinal chemistry. These collaborative problems encompass three themes: development of protein inhibitors as drugs, unravelling the influence of point mutations on protein structure, and deciphering how protein-protein interactions modulate activity and function.